Characterization of Haemophilus influenzae Strains with Non-Enzymatic Resistance to β-Lactam Antibiotics Caused by Mutations in the PBP3 Gene in the Czech Republic in 2010-2018

Abstract

The surveillance data on antibiotic resistance of Haemophilus influenzae have shown that strains with non-enzymatic resistance to β-lactam antibiotics have been on the rise in the Czech Republic over the last decade. This type of resistance is more difficult to detect than β-lactamase production. Analysis of 228 H. influenzae strains revealed that isolates with non-enzymatic resistance to β-lactams due to mutations in the ftsI gene could be reliably demonstrated by single run testing of susceptibility to amoxicillin/clavulanic acid (sensitivity of detection is 84.6%), cefuroxime (92.6%), ampicillin and penicillin (both 95.7%). Thirty-seven different amino acid substitution combinations were detected in the PBP3 protein at 23 positions (V329I, D350N, S357N, A368T, M377I, S385T, A388V, L389F, P393L, A437S, I449V, G490E, I491V, R501L, A502S, A502T, A502V, V511A, R517H, I519L, N526K, A530S, and T532S). The most common combination (35%) of amino acid substitutions was the combination D350N, M377I, A502V, N526K. Epidemiological typing does not indicate a clonal spread of a particular MLST type. Altogether there has been detected 74 STs. The most prevalent ST 1034 was associated mainly with a combination D350N, M377I, A502V, N526K. Clonal analysis revealed six clonal complexes (CCs) with the founder found, eight CCs without founder and 33 singletons.

Keywords: Haemophilus influenzae; ftsI; non-enzymatic resistance; β-lactam antibiotics.

Figure 1

The result of MLST-Hierarchical Clustering analysis (UPGMA method, Scale distance: 150; scale height: 16; cut off threshold: 3.49) of 228 strains of Haemophilus influenzae. Numbers are representing the names of STs. All figures were generated (computed and visualized) by PHYLOViZ 2.0 (www.phyloviz.net, accessed on 10 December 2020). The phylogenetic distance of individual STs indicates a wide origin and little interrelationship between the rPBP3 Group I, Group II, Group III and sPBP strains. * ST 1218 (n = 13, 5.7%) was the most frequent ST with R517H substitution (combination A502V, R517H, n = 11). ** ST 1034-the most represented ST in the whole set (n = 49, 21.5%) of strains, of which 48 had the most common combination of AA substitutions in the study: D350N, M377I, A502V, and N526K.